The overall goal of this research is to provide the means for systematically studying the genetic basis of virulence in Streptococcus mutans thus providing new knowledge for use in approaching the problem of caries control in man. A number of independently isolated strains of S. mutans have been found to harbor one of two unique plasmid systems: strains of human origin carry a 3.6 Mdal plasmid while those of rat origin contain a 2.2 Mdal plasmid with minor amounts of a 2.0 Mdal plasmids species. A portion of the work in this application is aimed at determining the function of these plasmids using comparative physiologic studies including the construction of isogenic plasmid-containing strains o S. sanguie by transformation. The major thrust of this research will be to develop one or more of these plasmids into a molecular cloning vehicle for use in studies aimed at exploring the genetic and biochemical basis of virulence in S. mutans. Preliminary work has led to the discovery of restriction endonucleases which cleave the 3.6 Mdal plasmid at one site thus facilitating the insertion on genetic information into this plasmid using recombinant DNA methodologies. Plasmid vector development will involve the identification of reference points on the plasmid DNA molecule using restriction site mapping and heterduplex analysis and may require the addition of selectable markers to the plasmid if no phenotypic trait is identified. A variety of approaches will be used in developing a genetic transformation system in S. mutans. Using such a transformation system and S. mutans plasmid vectors, we will clone chromosomal genes for exopolysaccharide production - a trait known to be closely associated with virulence. Biochemical examination of strains carrying cloned gene sequences will permit the assessment of a variety of genetic and physiologic parameters not currently amenable to study. This information may enable us to develop acariogenic strains of S. mutans which can supplant the cariogenic flora of the tooth. This work should provide a system for amplification of gene products which may be useful as antigens in caries immunoprophylaxis.